Project 3 is motivated by two concepts. First, CD4+ T lymphocytes (TCD4+), which recognize virus-derived peptides in the context of MHC class II molecules, are vital to the host in driving and potentiating adaptive responses and establishing immunological memory. Second, the interplay between pathogens and their natural hosts is exquisitely complex and experimental systems in which the virus and host have not coevolved risk missing crucial insights into these relationships. Despite this, there have been few studies of TCD4+ responses to a natural infection. Ectromelia virus, (ECTV) the mouse poxvirus, provides this opportunity in a highly relevant model featuring a broad range of susceptibilities. Though closely related, the much studied vaccinia virus (VACV) is not a mouse pathogen and preliminary results from all three labs have identified many fundamental differences between responses to ECTV and VACV. Thus, we expect that a systematic examination of the TCD4+ response to ECTV vs. VACV in resistant and susceptible mice will reveal insights with broad relevance. This highly integrated project has four independent but thematically connected aims. Aim 1 will define the peptide targets of TCD4+ responses, identify the processing pathways responsible for their generation, and ask whether peptide identity dictates TCD4+ phenotype. This work will be complemented in Aim 2 by imaging studies which will identify the relevant antigen presenting cells in vivo, determine how different types of antigen are presented in vivo, and examine the dynamics of TCD4+ activation following infection. In the third aim we will assess the role in protective immunity of peptides generated by a nonclassical but prevalent cytosolic processing pathway. Finally, published and preliminary data indicate that VACV interferes with class ll-restricted peptide presentation. In aim 4, we will assess the degree to which ECTV is committed to this endeavor. In addition to providing valuable information about the roles of TCD4+ in a natural infection, results from Project 3 integrate with those of the highly complementary Projects 1 and 2 to form a comprehensive picture of the ECTV/mouse dynamic. This picture will not only provide future directions for the program, but also insights into many other virus/host relationships.